The long-term aims of this project are to understand the role of neutral ceramidase (nCDase) in colon cancer pathogenesis, chemoprevention, and therapeutics. nCDase is a key, yet poorly studied, enzyme in regulating the interconversion of important bioactive molecules that include ceramides with an overall anti- tumor properties and sphingosine 1-phosphate with tumor-promoting properties. Our laboratory first identified, purified, and cloned the mammalian nCDase and studied its biochemical properties. In recent studies, we find that nCDase is enriched in the intestinal epithelium, and ongoing preliminary results show dramatic effects of deletion of nCDase on development of colon cancer in a mouse model of AOM (azoxymethane)-induced colonic adenocarcinoma. Additional recent studies are beginning to implicate this enzyme in the ss-catenin pathway. Based on the above, we propose the hypothesis that that nCDase is a 'synthetically lethal' target for transformed colon cells, especially those defined by abnormal activation of the ss-catenin pathway. We will employ chemical, biochemical, molecular, and in vivo studies to evaluate this hypothesis and to advance nCDase as a novel target. We will focus on the following specific aims: 1) Establish and define the role of NCDase in regulating colon cancer progression, by a) defining the in vivo role of nCDase in the development of colon cancer using the knock out mouse; b) determine the expression of nCDase in human colon cancer; and c) define the in vivo and cellular roles of nCDase in regulating cell growth and stress responses in colon cancer. 2) Determine the mechanisms underlying the prevention of colon cancer progression by disruption of nCDase. We will focus on a) determining the role of nCDase in regulating the ss-catenin pathway of colon cancer development in vivo and in cell culture models; b) defining the candidate bioactive lipid mediators of action of nCDase and roles of downstream targets; and c) defining mechanisms by which nCDase regulates the ss-catenin pathway. 3) Develop NCDase as a novel target for cancer therapy. Here we will a) develop small molecule inhibitors of nCDase based on initial hits; b) evaluate these inhibitors for their ability to inhibit nCDase in cells and the functional consequences; c) develop PK/PD studies; and d) Define the effects of these inhibitors in vivo. These results may allow us to define and establish nCDase as a novel and heretofore unappreciated major regulator of colon cancer progression. nCDase may emerge as a novel and effective target for rationally-based chemotherapy.